Ion channels are proteins that control the passive flux of selected ions through cell membranes by opening and closing (gating) their pores. Ion channels play a key role in many physiological processes, including integration of information in neurons, propagation of action potentials in nerve and muscle cells, synaptic transmission, and control of hormone secretion. This proposal seeks to continue work on investigating the fundamental mechanisms by which ion channels gate their pores. The focus of this research is on large conductance Ca2+-activated K+ (BK) channels from nerve and muscle. Although it is known that BK channels are activated by both increases in internal Ca2+ and depolarization of the membrane potential, the mechanisms by which this activation occurs is not clear. To work towards answering this question, the patch-clamp technique will be used to record ionic currents flowing through single BK channels in cultured rat skeletal muscle and also from cloned BK channels expressed in Xenopus oocytes and the HEK (human embryonic kidney) 293 cell line. The single-channel currents will then be analyzed with state-of-the-art techniques to determine the kinetic gating mechanisms of the channels. Such gating mechanisms will specify the numbers of states entered during gating, the transition pathways among the states, the rate constants for the transitions, and the changes in the rate constants produced by Ca2+i, voltage, and the beta subunit of the BK channel. Four specific aims will be carried out to resolve: 1) the mechanism by which the channels gate over the range of very low to very high Ca2+i; 2) the contributions of proposed primary and secondary Ca2+-binding sites to the gating; 3) the mechanism by which voltage activates the channels; and 4) the mechanism by which the beta subunit increases the Ca2+i sensitivity and alters the gating. An important step towards understanding how ion channels gate their pores and determining the contributions of ion channels to normal cellular function, as well as to identifying defective channels in disease processes, is to establish the kinetic gating mechanisms of the channels. This proposal will work towards this goal for BK channels.